Protective effect of interleukin-36 receptor antagonist on liver injury induced by concanavalin A in mice

Objective(s): Interleukin-36 receptor antagonist (IL-36Ra) is a new member of the IL-1 family that exhibits anti-inflammatory activity in a variety of inflammatory and immune diseases. Our purpose was to determine the effect of IL-36Ra on liver injury in a mouse hepatitis model induced by concanavalin A (ConA). Materials and Methods: Mice were treated with IL-36Ra DNA or pcDNA3.1 control plasmid using a hydrodynamic gene delivery approach. Results: Our data reveal that treatment with IL-36Ra decreased liver inflammation and serum level of aminotransferases. Furthermore, IL-36Ra reduced ConA-induced pro-inflammatory cytokines (interferon-γ, tumor necrosis factor-α, and IL-17A) production when compared to control plasmid. Conclusion: Our results demonstrated that IL-36Ra is a critical protector against ConA-induced liver injury.

Hepatitis is one of the most common diseases, which threatens human health and has a significant morbidity and mortality. Importantly, one of the most common causes of liver injury is autoimmune hepatitis (14). Concanavalin A (ConA)-induced hepatitis is a wellestablished murine model for human acute hepatitis that mimics human T-cell-mediated hepatitis condition in many respects (15), including markedly increased serum levels of aminotransaminase, pathological changes, and pro-inflammatory cytokine overproduction (16,17). Scheiermann et al. (18) recently reported that IL-36Ra can weaken the expression of the C-C chemokine CCL20 and impair recovery in the late phase of murine acetaminophen-induced liver injury. However, the role of IL-36Ra in ConA-induced liver injury is still unclear.
In this study, we investigated the significance of IL-36Ra in T cell-mediated hepatitis through a ConAinduced mouse hepatitis model.

Experimental animals
Male pathogen-free, BALB/c mice (6-8-weeks-old), weight ranging from 18 to 22 g, were obtained from Zhejiang University School of Medicine (Hangzhou, China). All experimental mice in this study were maintained and housed at the Animal Experimental Center of Ningbo University under standard laboratory conditions. Mice were provided standard water and food ad libitum in this study. The mice were allowed to adapt to the new environment for 5 days before the experiment, which was under a protocol approved by the Experimental Animal Ethics Committee of Ningbo University (SYDW20151001001).

Hydrodynamic plasmid injection
Plasmid DNA for IL-36Ra (constructed by our laboratory) was obtained from Escherichia coli using Plasmid Extraction kit (Tiangen Biotech, Beijing, China), and the plasmid DNA was dissolved in saline at a concentration of 50 μg/ml. Plasmid DNA was introduced into mice at a dose of 5 mg/kg using a hydrodynamicbased gene transfer approach (19) 24 hr before ConA (Sigma, St. Louis, MO., USA) injection. In our experiment, 1.2 mg plasmid DNA was diluted in 24 ml of saline for 12 mice (50 μg/ml) injection dosage. Plasmid pcDNA3.1-IL-36Ra-V5-His was injected into mice via the tail vein within 3 to 8 sec. The mice typically recovered from the injection within 5 to 10 min.

Detection of exogenous IL-36Ra in mouse liver
Livers were harvested from mice for studying IL-36Ra expression by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Mice were euthanized at 8, 24, 48, and 72 hr after the plasmid injection; each specified time point includes four mice. Another four mice were treated with an equal amount of saline and euthanized at 0 hr, as the normal control group.
For RT-PCR analysis, the total RNA was extracted from livers using RNAiso Plus Reagents (TaKaRa, Dalian, China) according to the manufacturer's instructions. The RNA was then used to synthesize the cDNA with a HiFiScript cDNA First Strand Synthesis Kit (CWBIO, Beijing, China). The PCR was performed with 2×EasyTaq PCR SuperMix, and the IL-36Ra forward and reverse primer sequences: 5′-CGGGGTACCATGGTCCTGAGTGGGGC-3′ and 5′-GCTCTAGAGTCACACTGCTGGAAGTAGAAGTC-3′ were used for detecting IL-36Ra mRNA. The PCR conditions were as follows: 30 sec at 94 °C for denaturation; 40 sec at 59 °C for annealing; 70 sec at 72 °C for extension; and 30 cycles. The PCR reaction was also performed with the housekeeping gene β-actin for normalization of results. The primer sequences for β-actin were 5'-TCCTGTGGGCATCCATGAAACT-3'; and 5'-GAAGCACTTGCGGTGCACGAT-3'. Western blot was used to detect IL-36Ra protein expression in the liver. Proteins were extracted from liver tissues using a RIPA Lysis Buffer (BeyoTime, Beijng, China), according to the manufacturer′s instructions. The proteins from representative mice were then subjected to western blot analysis. Membranes were incubated with 1:800 diluted anti-His-tag mouse primary antibodies (BeyoTime, Beijng, China) at 4 °C for 14 hr. The membranes were further washed with Tris-buffered saline Tween 20 (TBST) buffer three times, and then incubated with 1:5000 diluted horseradish peroxidase (HRP)conjugated goat anti-mouse IgG secondary antibodies for 45 min. Then, the protein bands were visualized by the enhanced chemiluminescence reaction method (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Treatment groups
Forty-eight mice were randomly divided into four groups, which include IL-36Ra-treated group (IL-36Ra/ ConA), pcDNA3.1-treated group (pcDNA3.1/ConA), saline-treated group (saline/ConA), and normal control group (saline only), with twelve mice included in each study group. The mice in the IL-36Ra-treated group and pcDNA3.1-treated group were intravenously injected with 2 ml of pcDNA3.1-IL-36Ra-V5-His (50 μg/ml) and 2 ml of pcDNA3.1 (50 μg/ml), respectively, while the saline-treated group and normal control group were injected with the same volume of saline. After 24 hr, the mice in groups (IL-36Ra/ConA, pcDNA3.1/ ConA, and saline/ConA) were challenged with 240 μl of ConA at a dose of 15 μg/g via tail vein injection (20), while the mice in the normal control group were administered an equal volume of saline. Blood samples were collected by cardiac puncture at 8 hr after ConA injection, stored at room temperature for 30 min, followed by centrifugation at 3,000 rpm at 4°C for 10 min, and then frozen at -40°C until further analysis. Initial study found that the distribution of ConA was specific for the liver (21). Therefore, mouse livers were harvested from euthanized mice at 8 and 24 hr after ConA administration for histopathological assay and analysis of cytokine mRNA expression.

Assay of serum aminotransaminase activity
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in mouse serum were measured by standard photometric methods using an ALT and AST diagnostic reagent kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Mouse liver histopathological examination
Mouse livers harvested at 8 hr and 24 hr were fixed in 10% neutral formalin for 48 hr. The tissues were treated with dehydrated ethanol, routinely embedded in paraffin wax and cut into 4 μm sections. Later, the sections were stained with hematoxylin-eosin (HE), and then examined for liver damage under an inverted light microscope at 10 × and 40 × magnification.

RT-PCR analysis of cytokine expression
Liver tissues were collected 8 hr after ConA challenge. For RT-PCR analysis, total RNAs were isolated from mouse liver using RNAiso plus regent according to the manufacturer's protocol. Total RNAs were treated with DNase and protease to remove genomic DNA and protein contamination, and cDNA was then synthesized from 4 μg of total RNA. Quantitative RT-PCR was performed in a 25 μl of reaction solution containing cDNA, primers and 2× EasyTaq PCR SuperMix. The mRNA levels of the proinflammatory cytokines IFN-γ, tumor necrosis factor alpha (TNF-α), and IL-17A were assessed by RT-PCR. Primer sequences (Generay Biotech, Shanghai, China) for each target gene are displayed in Table 1. The resulting PCR products for each target were analyzed in 1.2% agarose gels and stained with GelStain (Transgen, Beijing, China).

Measurement of serum pro-inflammatory cytokines
Serum samples were collected from mice euthanized at 8 hr after ConA treatment, and the concentrations of the inflammatory cytokines TNF-α, IFN-γ, and IL-17A in the serum were determined by enzyme-linked immunosorbent assays (ELISA) kit (ExCell Bio, Shanghai, China) according to the manufacturer's instruction.

IL-36Ra inhibits liver injury
Peng et al.

Statistical analysis
Data are reported as the mean±SEM. Differences between groups were evaluated by one-way ANOVA and Bonferroni statistical test. For each analysis, differences were considered statistically significant for P<0.05.

Exogenous IL-36Ra expression in mice
Mice were euthanized at 8, 24, 48, and 72 hr, respectively after hydrodynamic-based gene delivery. Liver tissues were acquired for RNA extraction and western blot. IL-36Ra mRNA was checked in the liver by semi-quantitative RT-PCR. RT-PCR detected a unique DNA fragment band of 468 bp corresponding to the expected size of IL-36Ra ( Figure 1A). IL-36Ra mRNA was present as early as 8 hr after gene delivery. The highest level was observed 24 hr after gene transfer, and IL-36Ra mRNA then showed a steady time-dependent decrease. However, the IL-36Ra mRNA was still detectable even after 72 hr of gene transfer.
To confirm the mRNA results, we then checked IL-36Ra protein expression in the liver using western blot. Western blot detected a unique band of approximately 17 kDa corresponding to the molecular weight of IL-36Ra ( Figure 1B). Importantly, the levels for IL-36Ra protein in the liver showed a similar trend to the mRNA levels measured previously. The highest protein levels were found at 24 hr of gene transfer.

IL-36Ra attenuates ConA-induced liver injury
The mice in the control groups (saline/ConA and pcDNA3.1/ConA) had a significantly increased serum concentration of ALT and AST after ConA injection. However, the serum ALT and AST levels in mice treated with the IL-36Ra plasmid (IL-36Ra/ConA) were significantly lower than mice of the control group (P <0.01) (Figure 2A). These results suggest that IL-36Ra markedly inhibited release of ALT and AST into the plasma of ConA-treated mice.
The pathological changes in the liver were examined by H&E staining of liver sections at 8 and 24 hr after ConA injection. Massive hepatocyte necrosis in both control groups (pcDNA3.1/ConA and saline/ConA) was observed. In sharp contrast, mice treated with IL-36Ra plasmid (IL-36Ra/ConA) displayed a markedly diminished area and extent of necrosis compared to the control groups ( Figure 2B). These pathological changes were consistent with changes in aminotransaminase level. Our results suggest that expression of exogenous IL-36Ra in the liver has a protective effect on ConAinduced hepatitis.  Table 1. Primer sequences used in this study for analysis of pro-inflammatory cytokines

IL-36Ra inhibited pro-inflammatory cytokines mRNA expression
ConA-induced hepatic injury was associated with changes in the expression of pro-inflammatory cytokines. To further confirm the above observations, we examined mRNA levels of cytokines in the mouse liver at 8 hr after ConA injection. We found that the expression of IFN-γ, TNF-α, and IL-17A mRNA in the liver was significantly increased in the ConA control groups. Importantly, at the same time, the expression of these cytokines in the IL-36Ra-treated group was significantly reduced relative to the ConA groups ( Figure 4).

Discussion
In the present study, we demonstrated that IL-36Ra was able to protect mice against ConA-induced hepatitis. The serum elevation of liver enzymes was significantly inhibited and liver necrosis was attenuated by IL-36Ra treatment. At the same time, IL-36Ra treatment significantly down-regulated the expression of IFN-γ, TNF-α, and IL-17A. These findings suggest that IL-36Ra is beneficial for protection against acute liver injury induced by ConA.
IL-36Ra is a new member of the IL-1 family. It is an anti-inflammatory cytokine together with IL-1Ra, IL-37 and IL-38, while the other seven members of IL-1 family are pro-inflammatory cytokines. Previous reports have shown that IL-1Ra (22), IL-37 (23) and IL-38 (24) play a protective role in ConA-induced liver injury. IL-36Ra has been identified as an inhibitory molecule for blocking the activations of NF-κB and MAPKs (11,12). To address whether IL-36Ra could be a therapeutic target for autoimmune hepatitis, we set out to investigate the significance of IL-36Ra in hepatic injury induced by ConA. The eukaryotic expression plasmid was introduced into mice by the hydrodynamic-based gene delivery procedure. Hydrodynamic injection is by far the most effective method for gene transfer into mouse liver. Another feature of this injection method is that it can transfer a large amount of plasmid solution into mice in 5-8 sec (25). In our experiment, the highest expression of IL-36Ra mRNA and protein was detected at 24 hr after IL-36Ra gene transfer, and there were no deaths caused by the procedure. In this study, IL-36Ra was thus successfully introduced into mice by hydrodynamicbased gene delivery procedure.
Serum ALT and AST levels and histopathology injury are widely used as conventional measurements for the evaluation of hepatic injury (26). Liver injury is usually accompanied by elevated serum ALT and AST levels, and aggravated liver histopathological damage. In this study, we measured liver injury according to the detection of serum concentrations of ALT and AST, and histopathological observations of liver injury using light microscopy. Our results showed that IL-36Ra delivery significantly inhibited liver necrosis, as judged by reduced serum ALT and AST concentrations and histopathological changes after ConA challenge.
ConA-induced hepatitis is a valid model of immunological hepatic injury. Experimental studies in animals have demonstrated that liver injury in ConAinduced hepatitis is associated with activated CD4 + T cells, macrophages, and natural killer T (NKT) cells that cause hepatocyte injury by producing lots of proinflammatory cytokines, particularly IFN-γ and TNF-α (15,21,(26)(27)(28). Notably, antibodies against IFN-γ and TNF-α can attenuate ConA-induced liver injury in mice (29,30). IFN-γ is produced by immune cells such as T cells, NK cells, and NKT cells. Aberrant IFN-γ expression has been associated with numerous inflammatory and autoimmune diseases, including autoimmune hepatitis   (30). The inflammatory pathway regulated by IFN-γ in the liver also plays a crucial role in immune responses in autoimmune hepatitis (31). IL-17A has been demonstrated to be essential for the development of various autoimmune diseases (32). It is mainly secreted by CD4 + T cells, NKT cells, and gamma delta T (γδT) cells, and the liver contains all these cell types (33). Previous studies have shown that IL-17A may exacerbate liver injury by recruiting neutrophils (34,35). Vigne et al. (36,37) showed that IL-36β promotes IFN-γ production by CD4 + T cells and cultured splenocytes, and these stimulatory effects were antagonized by IL-36Ra. Similarly, Gresnigt et al. (38) found that IL-36Ra reduces aspergillus-induced IL-17 and IFN-γ production. Consistent with those published data, our experiments showed that IL-36Ra treatment significantly reduced TNF-α, IFN-γ and IL-17A expression in ConA-induced liver injury in mice.

Conclusion
Our present study revealed that IL-36Ra treatment significantly attenuated ConA-induced liver injury. Hepatic necrosis was significantly inhibited in mice, and serum levels of ALT and AST were reduced accordingly. The protective role of IL-36Ra was associated with the reduced expression of the pro-inflammatory cytokines TNF-α, IFN-γ, and IL-17A. Our results suggest that IL-36Ra may serve as a novel drug target for the treatment of hepatitis.